I. Field of the Invention
This invention relates to the formation of derivatives of organic compounds and, more particularly, to such derivatization carried out as a step during highly sensitive analysis or determination of various organic compounds available in very dilute aqueous solutions.
II. Description of the Prior Art
In recent years, techniques have been developed for the analysis or determination of organic compounds present in extremely small quantities or at very low concentrations. For example, by combining gas chromatography with electron capture detection or mass spectrometry operating in the negative ion chemical ionization mode, organic chemicals present in solution at concentrations of micrograms per ml or less can be analysed. Such techniques are extremely useful for the analysis of so-called "biological matrices", i.e. aqueous fluids obtained from living organisms, such as plasma, serum, urine etc. from man and animals and fluid media from microorganisms. A great deal of information can be learned about the organisms if the organic chemicals present in such fluids, often in only trace quantities, can be analysed or determined.
A characteristic of such highly sensitive techniques is that they require the presence of certain specific groups in the molecule, e.g. chromophores, fluorophores or electrophores. For example, electron capture detection requires the presence of an electrophore (a group capable of capturing electrons), e.g. a group containing halogen atoms covalently bound to carbon. Unfortunately many organic compounds of interest for analysis, particularly those from biological matrices, do not possess such groups and must be converted to suitable derivatives as a preliminary step. In some cases it is also necessary to convert unstable compounds to stable derivatives.
As an example, organic chemicals containing carboxylic acid groups may be converted to trichloroethyl or pentafluorobenzyl esters, and compounds containing amino or hydroxyl groups may be converted to trifluoroacyl or pentafluorobenzyl derivatives. This introduces electrophores suitable for highly sensitive analysis and makes the compounds more suitable for purification or separation by gas chromatography.
Derivatization of organic compounds occurring in biological matrices has followed two general approaches in the past. In the first approach, the organic compound to be analysed or determined (the analyte) is first isolated from the biological matrix, concentrated, derivatized by reaction with a derivatizing agent in the presence of a catalyst and then purified. In the second approach, the analyte is simultaneously extracted and derivatized by the use of a plurality of liquid phases and a phase transfer catalyst, followed by purification.
These two conventional approaches have significant disadvantages, the main ones being that they are labour intensive and do not easily lend themselves to automation. Both approaches involve liquid/liquid phase separations and evaporation steps that are not easy to carry out automatically. Moreover, phase transfer catalysts tend to be isolated along with the resulting derivative and the presence of such catalysts interferes with the subsequent analysis. Further, the catalysts themselves tend to be expensive and difficult to use.
Various partial solutions have been found to resolve these problems. Solid/liquid extraction methods have been developed to permit automated isolation of an underivatized analyte. However, these methods still require a separate derivitization step to be carried out on the analyte prior to analysis. In the case of phase transfer catalysts, methods have been developed to remove traces of the catalysts isolated with the derivative, but these methods require extra steps in a procedure designed to simplify sample preparation. Phase transfer catalysts have also been covalently linked to insoluble supports, such as polystyrene or silica gel, and the resulting mechanism of triphasic catalysts has been extensively studied, but these techniques have not as yet been applied to analytical chemistry.
Reference is made to "Mass Fragmentographic Assay for 11-hydroxy-.DELTA..sup.9 -tetrahydro-cannabinol from Plasma" by J. M. Rosenfeld and V. Y. Taguchi, Analytical Chemistry, Vol. 48, pp. 726 to 792, April 1976, as an example showing a conventional derivatization technique.
An object of the invention is therefore to overcome at least some of the disadvantages of the prior art by providing a simplified derivatization technique that can, if desired, be carried out automatically.